Abstract
To evaluate its potential as a ligand discovery tool, we compare a newly developed 1D protein-observed fluorine NMR (PrOF NMR) screening method with the well-characterized ligand-observed 1H CPMG NMR screen. We selected the first bromodomain of Brd4 as a model system to benchmark PrOF NMR because of the high ligandability of Brd4 and the need for small molecule inhibitors of related epigenetic regulatory proteins. We compare the two methods' hit sensitivity, triaging ability, experiment speed, material consumption, and the potential for false positives and negatives. To this end, we screened 930 fragment molecules against Brd4 in mixtures of five and followed up these studies with mixture deconvolution and affinity characterization of the top hits. In selected examples, we also compare the environmental responsiveness of the 19F chemical shift to 1H in 1D-protein observed 1H NMR experiments. To address concerns of perturbations from fluorine incorporation, ligand binding trends and affinities were verified via thermal shift assays and isothermal titration calorimetry. We conclude that for the protein understudy here, PrOF NMR and 1H CPMG have similar sensitivity, with both being effective tools for ligand discovery. In cases where an unlabeled protein can be used, 1D protein-observed 1H NMR may also be effective; however, the 19F chemical shift remains significantly more responsive.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 3154-3164 |
| Number of pages | 11 |
| Journal | ACS Chemical Biology |
| Volume | 11 |
| Issue number | 11 |
| DOIs | |
| State | Published - Nov 18 2016 |
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Protein-observed fluorine NMR is a complementary ligand discovery method to 1H CPMG ligand-observed NMR. / Urick, Andrew K.; Calle, Luis Pablo; Espinosa, Juan F.; Hu, Haitao; Pomerantz, William C.K.
In: ACS Chemical Biology, Vol. 11, No. 11, 18.11.2016, p. 3154-3164.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Protein-observed fluorine NMR is a complementary ligand discovery method to 1H CPMG ligand-observed NMR
AU - Urick, Andrew K.
AU - Calle, Luis Pablo
AU - Espinosa, Juan F.
AU - Hu, Haitao
AU - Pomerantz, William C.K.
PY - 2016/11/18
Y1 - 2016/11/18
N2 - To evaluate its potential as a ligand discovery tool, we compare a newly developed 1D protein-observed fluorine NMR (PrOF NMR) screening method with the well-characterized ligand-observed 1H CPMG NMR screen. We selected the first bromodomain of Brd4 as a model system to benchmark PrOF NMR because of the high ligandability of Brd4 and the need for small molecule inhibitors of related epigenetic regulatory proteins. We compare the two methods' hit sensitivity, triaging ability, experiment speed, material consumption, and the potential for false positives and negatives. To this end, we screened 930 fragment molecules against Brd4 in mixtures of five and followed up these studies with mixture deconvolution and affinity characterization of the top hits. In selected examples, we also compare the environmental responsiveness of the 19F chemical shift to 1H in 1D-protein observed 1H NMR experiments. To address concerns of perturbations from fluorine incorporation, ligand binding trends and affinities were verified via thermal shift assays and isothermal titration calorimetry. We conclude that for the protein understudy here, PrOF NMR and 1H CPMG have similar sensitivity, with both being effective tools for ligand discovery. In cases where an unlabeled protein can be used, 1D protein-observed 1H NMR may also be effective; however, the 19F chemical shift remains significantly more responsive.
AB - To evaluate its potential as a ligand discovery tool, we compare a newly developed 1D protein-observed fluorine NMR (PrOF NMR) screening method with the well-characterized ligand-observed 1H CPMG NMR screen. We selected the first bromodomain of Brd4 as a model system to benchmark PrOF NMR because of the high ligandability of Brd4 and the need for small molecule inhibitors of related epigenetic regulatory proteins. We compare the two methods' hit sensitivity, triaging ability, experiment speed, material consumption, and the potential for false positives and negatives. To this end, we screened 930 fragment molecules against Brd4 in mixtures of five and followed up these studies with mixture deconvolution and affinity characterization of the top hits. In selected examples, we also compare the environmental responsiveness of the 19F chemical shift to 1H in 1D-protein observed 1H NMR experiments. To address concerns of perturbations from fluorine incorporation, ligand binding trends and affinities were verified via thermal shift assays and isothermal titration calorimetry. We conclude that for the protein understudy here, PrOF NMR and 1H CPMG have similar sensitivity, with both being effective tools for ligand discovery. In cases where an unlabeled protein can be used, 1D protein-observed 1H NMR may also be effective; however, the 19F chemical shift remains significantly more responsive.
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U2 - 10.1021/acschembio.6b00730
DO - 10.1021/acschembio.6b00730
M3 - Article
C2 - 27627661
AN - SCOPUS:84996946632
VL - 11
SP - 3154
EP - 3164
JO - ACS Chemical Biology
JF - ACS Chemical Biology
SN - 1554-8929
IS - 11
ER -